Publications

• Glucose permease of the bacterial phosphotransferase system. Gene cloning, overproduction, and amino acid sequence of enzyme IIGlc.

  Erni B., Zanolari B.

  The glucose-permease (IIGlc) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It also functions as a receptor for bacterial chemotaxis. The structural gene of the permease, ptsG, has been cloned on a multico ... >> More

  The glucose-permease (IIGlc) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It also functions as a receptor for bacterial chemotaxis. The structural gene of the permease, ptsG, has been cloned on a multicopy plasmid, and transformants constitutively overproducing the protein 10-15 times over wild-type level have been isolated. Overproduction is slightly inhibited if transformants are grown in a glucose-containing medium. The complete amino acid sequence of the glucose-permease is deduced from the nucleotide sequence. It consists of 477 residues and is moderately hydrophobic. A comparison of the glucose-permease with the mannitol-permease (Lee, C. A., and Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 10761-10767) does not reveal any obvious homology at the level of amino acid sequence. << Less

  J. Biol. Chem. 261:16398-16403(1986) [PubMed] [EuropePMC]

• Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a glucose-specific phosphocarrier protein (IIIGlc) from Salmonella typhimurium.

  Meadow N.D., Roseman S.

  The phosphocarrier protein, IIIGlc, of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was purified to homogeneity by two methods. The first method utilized ion exchange and gel filtration chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis, and required se ... >> More

  The phosphocarrier protein, IIIGlc, of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was purified to homogeneity by two methods. The first method utilized ion exchange and gel filtration chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis, and required several weeks for completion. The second method utilized and antibody affinity column plus two additional steps and could be completed in a few days. By both procedures, two forms of IIIGlc were isolated, which were called IIIGlc Slow and IIIGlc Fast on the basis of their relative mobilities in polyacrylamide gels. IIIGlc Fast is derived from IIIGlc Slow by cleavage of the seven NH2-terminal amino acids from the latter protein. Both IIIGlc Slow and IIIGlc Fast have Mr approximately 20,000; neither protein contains cysteine, tyrosine, or tryptophan. IIIGlc Slow is very stable to heat; only 50% of its sugar phosphorylating activity is lost after 1 h at 100 degrees C. The phosphoryl group in IIIGlc Slow appears to be linked to a histidinyl residue. Direct transfer of the phosphoryl group from HPr (the histidine-containing phosphocarrier protein of the PTS) to IIIGlc slow was demonstrated as well as the reverse reaction. In addition, phospho-IIIGlc Slow served as a phosphoryl donor to methyl alpha-glucoside (or glucose) in the absence of all other PTS components except the partially purified integral membrane protein specific for this sugar, II-BGlc. The loss of the seven amino acids from IIIGlc Slow (giving IIIGlc Fast) leads to a marked alteration in the kinetic properties of the protein in the phosphotransferase system. IIIGlc Slow accepts 1 mol of phosphate from phosphoenolpyruvate via Enzyme I and HPr (the histidine-containing phosphocarrier protein) and participates in the phosphorylation of glucose or methyl alpha-D-glucoside. IIIGlc Fast also accepts 1 mol of phosphate, but phospho-IIIGlc Fast is only 2-3% as active as phospho-IIIGlc Slow in the phosphorylation of sugar. IIIGlc Fast is found only in trace quantities in living cells, and may play a role in the regulation of non-PTS sugar transport systems. << Less

  J. Biol. Chem. 257:14526-14537(1982) [PubMed] [EuropePMC]

• Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system -- two highly similar glucose permeases in Staphylococcus carnosus with different glucoside specificity: protein engineering in vivo?

  Christiansen I., Hengstenberg W.

  Previous sequence analysis of the glucose-specific PTS gene locus from Staphylococcus carnosus revealed the unexpected finding of two adjacent, highly similar ORFs, glcA and glcB, each encoding a glucose-specific membrane permease EIICBA(Glc). glcA and glcB show 73% identity at the nucleotide leve ... >> More

  Previous sequence analysis of the glucose-specific PTS gene locus from Staphylococcus carnosus revealed the unexpected finding of two adjacent, highly similar ORFs, glcA and glcB, each encoding a glucose-specific membrane permease EIICBA(Glc). glcA and glcB show 73% identity at the nucleotide level and glcB is located 131 bp downstream from glcA. Each of the genes is flanked by putative regulatory elements such as a termination stem-loop, promoter and ribosome-binding site, suggesting independent regulation. The finding of putative cis-active operator sequences, CRE (catabolite-responsive elements) suggests additional regulation by carbon catabolite repression. As described previously by the authors, both genes can be expressed in Escherichia coli under control of their own promoters. Two putative promoters are located upstream of glcA, and both were found to initiate transcription in E. coli. Although the two permeases EIICBA(Glc)1 and EIICBA(Glc)2 show 69% identity at the protein level, and despite the common primary substrate glucose, they have different specificities towards glucosides as substrate. EIICBA(Glc)1 phosphorylates glucose in a PEP-dependent reaction with a Km of 12 microM; the reaction can be inhibited by 2-deoxyglucose and methyl beta-D-glucoside. EIICBA(Glc)2 phosphorylates glucose with a Km of 19 microM and this reaction is inhibited by methyl alpha-D-glucoside, methyl beta-D-glucoside, p-nitrophenyl alpha-D-glucoside, o-nitrophenyl beta-D-glucoside and salicin, but unlike other glucose permeases, including EIICBA(Glc)1, not by 2-deoxyglucose. Natural mono- or disaccharides, such as mannose or N-acetylglucosamine, that are transported by other glucose transporters are not phosphorylated by either EIICBA(Glc)1 nor EIICBA(Glc)2, indicating a high specificity for glucose. Together, these findings support the suggestion of evolutionary development of different members of a protein family, by gene duplication and subsequent differentiation. C-terminal fusion of a histidine hexapeptide to both gene products did not affect the activity of the enzymes and allowed their purification by Ni2+-NTA affinity chromatography after expression in a ptsG (EIICB(Glc)) deletion mutant of E. coli. Upstream of glcA, the 3' end of a further ORF encoding 138 amino acid residues of a putative antiterminator of the BglG family was found, as well as a putative target DNA sequence (RAT), which indicates a further regulation by glucose specific antitermination. << Less

  Microbiology 145:2881-2889(1999) [PubMed] [EuropePMC]

• Glucose permease of Escherichia coli. The effect of cysteine to serine mutations on the function, stability, and regulation of transport and phosphorylation.

  Nuoffer C., Zanolari B., Erni B.

  The glucose permease (IIGlc/IIIGlc complex) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It contains 3 cysteine residues, of which Cys-204 and Cys-326 are localized in the hydrophobic part and Cys-421 in ... >> More

  The glucose permease (IIGlc/IIIGlc complex) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It contains 3 cysteine residues, of which Cys-204 and Cys-326 are localized in the hydrophobic part and Cys-421 in the hydrophilic part of the IIGlc subunit. The cysteines were replaced, one at a time, by serines, and the effect of these mutations on stability, regulation, and catalytic properties of IIGlc was investigated in vivo and in vitro. Cys-204 and Cys-326 are not required for catalytic function and are not involved in the membrane potential-dependent regulation of IIGlc activity (Robillard, G. T., and Konings, W. N. (1982) Eur. J. Biochem. 127, 597-604). Replacement of these cysteines by serines results, however, in reduced stability of IIGlc in vivo (C204S) and in vitro (C204S and C326S), indicating that these substitutions in a hydrophobic environment can destabilize the protein structure. Cys-421 is absolutely required for transport and phosphorylation of glucose. C421S can neither be phosphorylated by phospho-IIIGlc nor catalyze the phosphoryl exchange between [14C] glucose and glucose 6-phosphate at equilibrium. C421S does not interfere with the activity of simultaneously expressed wild-type IIGlc. Unexpectedly C421S and wild-type IIGlc support growth on maltose of Escherichia coli ZSC112L (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199), a strain which otherwise does not grow on this disaccharide as the only carbon source. C421S appears to facilitate the efflux of a growth inhibiting intermediate (glucose?) of maltose. Wild-type IIGlc catalyzes the intracellular phosphorylation of glucose derived from maltose. It is concluded that the cytoplasmic domain of IIGlc interacts with IIIGlc, the cytoplasmic subunit of the glucose permease, and also participates in phosphorylation of glucose, and that phosphorylation occurs independently of transport, although transport of glucose by wild-type IIGlc cannot occur without concomitant phosphorylation. << Less

  J. Biol. Chem. 263:6647-6655(1988) [PubMed] [EuropePMC]

• Sugar transport by the bacterial phosphotransferase system. The glucose receptors of the Salmonella typhimurium phosphotransferase system.

  Stock J.B., Waygood E.B., Meadow N.D., Postma P.W., Roseman S.

  We have previously reported that glucose can be phosphorylated by phospho-HPr and two sugar-specific pairs of proteins of the Escherichia coli and Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Each of the sugar-specific complexes comprises two proteins, lipid, and d ... >> More

  We have previously reported that glucose can be phosphorylated by phospho-HPr and two sugar-specific pairs of proteins of the Escherichia coli and Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Each of the sugar-specific complexes comprises two proteins, lipid, and divalent cation, and each is present in membranes isolated from wild type cells. For reasons described in this report, one of the complexes is designated IIGlc and the other IIMan. The IIMan complex has previously been separated into its protein components, II-A and II-B (Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 1407-1418), while the accompanying reports describe dissociation of the IIGlc complex into its components, IIIGlc and II-BGlc. Curtis and Epstein (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199) first showed that there are two phosphotransferase systems in whole cells responsible for glucose uptake and obtained the respective mutants, now designated ptsG and ptsM. The present studies provide kinetic conditions for assaying each activity separately (in vivo and in vitro), when both are present in the same membrane preparation. The IIGlc system is responsible for the uptake and phosphorylation of glucose and methyl alpha-glucoside, whereas the IIMan system is less specific and utilizes glucose, mannose, and 2-deoxyglucose. With high sugar concentrations in vitro, IIMan is also capable of phosphorylating methyl alpha-glucoside, fructose, and N-acetylmannosamine, while IIGlc phosphorylates fructose and mannose. The in vivo transport results were qualitatively consistent with the in vitro phosphorylation results, and several of the kinetic parameters also showed good quantitative agreement. The levels of the two activities depended on the growth conditions. In addition, transport studies showed that initial uptake rates of methyl alpha-glucoside and steady state levels of this analogue depended on the energy state of the cells and that these two parameters did not necessarily change in the same direction when metabolic inhibitors were used. A series of E. coli and S. typhimurium mutants were characterized both with respect to their ability to transport the glucose analogues and to phosphorylate them in vitro. The original mutants of Curtis and Epstein, ptsG and ptsM, were found to be defective in II-BGlc and the IIMan complex, respectively. << Less

  J. Biol. Chem. 257:14543-14552(1982) [PubMed] [EuropePMC]